20(S) camptothecin glycoconjugates

ABSTRACT

The present invention relates to glycoconjugates of 20(S)-camptothecin, in which a 3-O-methylated beta-L-fucose unit is linked to the 20-hydroxyl group of a camptothecin derivative via a thiourea-modified peptide spacer. The invention furthermore relates to processes for the preparation of the compounds according to the invention and to their use as medicaments, in particular in connection with oncoses.

This application is a 371 of PCT/EP98/02620, filed May 4, 1998.

The present invention relates to glycoconjugates of 20(S)-camptothecin,in which a 3-O-methylated β-L-fucose unit is linked to the 20-hydroxylgroup of a camptothecin derivative via thiourea-modified peptidespacers. The invention furthermnore relates to processes for thepreparation of the compounds according to the invention and to their useas medicaments, in particular in connection with oncoses.

20-(S)-Camptothecin is a pentacyclic alkaloid which was isolated in 1966by Wall et al. (J. Am. Chem. Soc. 88, 3888 (1966)). It has a highantitumour active potential in numerous in vitro and in vivo tests.Unfortunately, however, the promising potential failed to be realized inthe clinic because of toxicity and solubility problems.

By opening of the E ring lactone and formation of the sodium salt, awater-soluble compound was obtained which is in a pH-dependentequilibrium with the ring-closed form. Here too, clinical studies havebeen unsuccessful until now.

Approximately 20 years later, it was found that the biological activityis to be attributed to an enzyme inhibition of the topoisomerase I.Since then, the research activities have been increased again in orderto find camptothecin derivatives which are more compatible and active invivo.

To improve the water-solubility, salts of A ring- and B ring-modifiedcamptothecin derivatives and of 20-O-acyl derivatives having ionizablegroups have been described (Vishnuvajjala et al. U.S. Pat. No.4,943,579). The latter prodrug concept was later also applied tomodified camptothecin derivatives (Wani et al. WO 9602546). In vivo,however, the 20-O-acyl prodrugs described have a very short half-lifeand are very rapidly cleaved to give the parent structure.

WO 9631532 A1 describes sugar-modified cytostatics in which the linkageof various cytotoxic or cytostatically active compounds to, for example,regioselectively modified carbohydrate units via specific spacers leadto an improvement in the tumour selectivity. From the combinations ofcarbohydrate, spacer and active compound widely described there, we thensurprisingly found that the linkage of β-L-fucose units modified in the3-position via a thiourea-modified peptide spacer consisting of asterically demanding non-polar side chain-containing and a basic sidechain-containing amino acid on the 20-hydroxyl group of20(S)-camptothecin leads to very particularly preferred conjugateshaving the following properties:

By means of the ester-like linkage of the carrier radical to the20-hydroxyl group, the lactone ring in the camptothecin moiety, which isimportant for the action, is stabilized.

By means of the special conformation of the dipeptide spacers, theconjugates in extracellular medium and in blood have a stability whichis again markedly improved in comparison with similar conjugates havingother spacers previously described in WO 9631532. In particular, theconjugates according to the invention are more stable than the 20-O-acylprodrugs of camptothecin described in U.S. Pat. No. 4,943,579.

The conjugates according to the invention have better water solubilityin comparison with similar conjugates from WO 9631532.

In vitro, the conjugates according to the invention have a high activityagainst tumour cell lines and tumour xenografts.

In vivo, the conjugates according to the invention have excellenttherapeutic activity over several dose stages against various tumoursafter i.v. administration.

Compared with the underlying toxophore they have a markedly highertolerability and tumour selectivity, in particular with respect to bonemarrow toxicity.

The invention relates to compounds of the formula (I)

in which

R¹ represents a sterically demanding non-polar side chain of an aminoacid and

R² represents a basic side chain of an amino acid and their salts,stereoisomers and stereoisomer mixtures.

Preferred compounds of the formula (I) are those

in which

R¹ is a branched alkyl radical having up to 4 carbon atoms and

R² is a radical of the formula —(CH₂)_(n)—R3, where

 and

n is a number 1 to 4.

Particularly preferred compounds of the general formula (I) are those inwhich

R¹ is a branched alkyl radical of the formula

 and

R² is a radical of the formula

The camptothecin unit can be present in the 20(R) or in the 20(S)configuration or as a mixture of these two stereoisomeric forms. The20(S) configuration is preferred.

The amino acids can occur in the L or in the D configuration oralternatively as a mixture of D and L form.

The term “amino acids” in particular designates the α-amino acidsoccurring in nature, but moreover also comprises their homologues,isomers and derivatives. As an example of isomers; enantiomers may bementioned. Derivatives can be, for example, amino acids provided withprotective groups.

Amino acids having “sterically demanding” side chains are understood asmeaning those amino acids whose side chain has a branching in the β- orγ-position; examples which may be mentioned are valine and isoleucine orleucine.

Typical examples of amino acids having non-polar side chains which maybe mentioned are:

alanine, valine, leucine, isoleucine, proline, tryptophan,phenylalanine, methionine.

Typical examples of amino acids having basic side chains which may bementioned are:

lysine, arginine, histidine, ornithine, diaminobutyric acid.

The compounds according to the invention are preferably present in theform of their salts. In general, salts with organic or inorganic acidsmay be mentioned here.

The acids which can be adducted preferably include hydrohalic acids,such as, for example, hydrochloric acid and hydrobromic acid, inparticular hydrochloric acid, furthermore phosphoric acid, nitric acid,sulphuric acid, mono- and bifunctional carboxylic acids andhydroxycarboxylic acids, such as, for example, acetic acid,trifluoroacetic acid, maleic acid, malonic acid, oxalic acid, gluconicacid, succinic acid, fumaric acid, tartaric acid, citric acid, salicylicacid, sorbic acid and lactic acid and also sulphonic acids, such as, forexample, p-toluenesulphonic acid, 1,5-naphthalenedisulphonic acid orcamphorsulphonic acid.

The glycoconjugates according to the invention can be prepared, forexample, by linkage of 20(S)-camptothecin to activated carboxylcomponents, which for their part can be moieties of protected aminoacids, peptides or carbohydrate-modified peptides.

Preferably, the synthesis of the glycoconjugate takes placesequentially, beginning with the acylation of 20(S)-camptothecin with anN-protected carboxyl-activated unit of a non-polar sterically demandingamino acid in a suitable solvent, if appropriate in the presence of abase, according to customary methods. The amino protective group is thenremoved selectively by means of known methods. A unit of a basic aminoacid, which, if necessary, is suitably protected is then linked andsubsequently, if appropriate with retention of the side chain protectivegroup, deblocked at the α-amino function. In the key step, the linkageto the carbohydrate radical is carried out by conversion ofp-aminophenyl-3-O-methyl-β-L-fucopyranoside into the correspondingisothiocyanate and subsequent linkage to the deblocked α-amino group ofthe peptidyl camptothecin. Side chain protective groups which may stillbe present are detached and the free amino group is optionally convertedinto a suitable ammonium salt.

The invention thus furthermore relates to a process for the preparationof the glycoconjugates of the formula (I)

in which

R¹ represents a sterically demanding non-polar side chain of an aminoacid and

R² represents a basic side chain of an amino acid,

or of their salts, characterized in that the isothiocyanate of theformula (II)

is reacted with the peptidyl-camptothecin, optionally bearing aprotective group in the side chain, of the formula (III)

in which

R¹ has the abovementioned meaning and

R^(2′) has the meaning of the abovementioned basic radical R², whichmoreover can carry a protective group customary in peptide chemistry onthe basic group

to give the glycoconjugate of the formula (IV)

in which

R¹ and R^(2′) have the meanings indicated above,

the side chain amino protective group which may be present is removedaccording to customary methods and the compound obtained is optionallyconverted into the desired salt.

Another sequence of reaction steps in the synthesis of the targetcompound is also conceivable. Thus, according to a likewise preferredvariant, the p-isothiocyanatophenyl-3-O-methyl-β-L-fucoside can also belinked first with the optionally suitably protected terminal basic aminoacid, and this unit can then be reacted with the free amino group of theamino acid conjugate of 20(S)-camptothecin and the non-polar, stericallydemanding amino acid. Side chain protective groups which may be presentare detached and the free amino group is optionally converted into asuitable ammonium salt.

The invention therefore further relates to an alternative process forthe preparation of compounds of the general formula (I) or of theirsalts, characterized in that the isothiocyanate of the formula (II)

is reacted with an optionally suitably protected terminal basic aminoacid of the formula (V)

in which R^(2′) represents a basic side chain of an amino acid whosebasic group can be protected,

to give an amino acid conjugate of the formula (VI)

in which R^(2′) has the meaning indicated above,

this is then reacted with amino acid conjugates of the formula (VII)

in which R¹ has the meaning indicated above,

the side chain protective group is removed and the compounds areoptionally converted into a suitable salt.

Diastereomer mixtures can be formed, in particular after linkage of thefirst amino acid to camptothecin. Pure diastereomers of the compoundsaccording to the invention can be prepared by the process indicatedabove, for example, by separating the diastereomers in a suitable mannerafter linkage of the first amino acid unit to the camptothecin andsubsequent protective group removal. The diastereomerically pure targetcompound can be prepared from a diastereomerically pure intermediatecompound by the route indicated above.

The diastereomer mixture of the target compound can also be separatedinto the individual diastereomers in a customary manner.

The reactions can be carried out under various pressure and temperatureconditions, for example 0.5 to 2 bar, and −30 to +100° C., in suitablesolvents such as dimethylformamide (DMF), tetrahydrofuran (THF),dichloromethane, chloroform, lower alcohols, acetonitrile, dioxane,water or in mixtures of the solvents mentioned. As a rule, reactions inDMF, dichloromethane or THF/dichloromethane at room temperature andnormal pressure are preferred.

For the activation of the carboxyl groups, possible coupling reagentsare those known in peptide chemistry such as described, for example, inJakubke/Jeschkeit: Aminosäuren, Peptide, Proteine (Amino Acids,Peptides, Proteins); Verlag Chemie 1982 or Tetrahedr. Lett. 34, 6705(1993). N-Carboxylic anhydrides, acid chlorides or mixed anhydrides, forexample, are preferred.

Furthermore suitable for the activation of the carboxyl groups is theformation of adducts with carbodiumides, e.g. N,N′-diethyl-,N,N′-diisopropyl-, N,N′-dicyclohexylcarbodiimide,N-(3-dimethylaminopropyl)-N′-ethyl-carbodiumide hydrochloride,N-cyclohexyl-N′-(2-morpholinoethyl)-carbodiimidemetho-p-toluenesulphonate, or carbonyl compounds such ascarbonyldiimidazole, or 1,2-oxazolium compounds such as2-ethyl-5-phenyl-1,2-oxazolium-3-sulphate or2-tert-butyl-5-methyl-isoxazolium perchlorate, or acylamino compoundssuch as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, orpropanephosphonic anhydride, or isobutyl chloroformate, orbenzotriazolyloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate,1-hydroxybenzotriazole esters or N-hydroxysuccinimide esters.Furthermore, the amino acid components can also be employed in the formof a Leuch' anhydride.

Bases employed can be, for example, triethylamine,ethyl-diisopropylamine, pyridine, N,N-dimethylaminopyridine or others.

Protective groups employed for third functions of the amino acids can bethe protective groups known in peptide chemistry, for example of theurethane, alkyl, acyl, ester or amide type.

Amino protective groups in the context of the invention are thecustomary amino protective groups used in peptide chemistry.

These preferably include: benzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,2,4-dimethoxybenzyloxycarbonyt, 4-methoxybenzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl,tert-butoxycarbonyl (Boc), allyloxycarbonyl, vinyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl, phthaloyl,2,2,2-trichloroethoxycarbonyl, 2,2,2-trichloro-tert-butoxycarbonyl,menthyloxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl(Fmoc), formyl, acetyl, propionyl, pivaloyl, 2-chloroacetyl,2-bromoacetyl, 2,2,2-trifluoroacetyl, 2,2,2-trichloroacetyl, benzoyl,benzyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, phthalimido,isovaleroyl or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl,4-nitrophenyl or 2-nitrophenylsulphenyl. The Fmoc group and the Bocgroup are particularly preferred.

Preferred carboxyl protective groups are linear or branched C₁- toC₄-alkyl esters.

The removal of protective groups in appropriate reaction steps can becarried out, for example, by reaction of acid or base,hydrogenolytically or reductively in another manner.

Both in vitro and in vivo, the glycoconjugates according to theinvention have a surprisingly strong antitumour activity against varioustumours, in particular lung, breast, pancreas, melanoma and largeintestine tumours, combined with a great selectivity againstnon-malignant cells.

They are therefore suitable for the treatment of oncoses, to be specificboth in human and in veterinary medicine.

The present invention includes pharmaceutical preparations which, inaddition to non-toxic, inert pharmaceutically suitable excipients,contain one or more compounds according to the invention or whichconsist of one or more active compounds according to the invention, andprocesses for the production of these preparations.

The active compounds can optionally be present in one or more of theexcipients indicated above and also in microencapsulated form.

The therapeutically active compounds should be present in theabovementioned pharmaceutical preparations in a concentration fromapproximately 0.1 to 99.5, preferably from approximately 0.5 to 95%, byweight of the total mixture.

Apart from the compound according to the invention, the abovementionedpharmaceutical preparations can also contain further pharmaceuticalactive compounds.

In general, it has proven advantageous both in human and in veterinarymedicine to administer the active compound according to the invention intotal amounts of approximately 0.5 to approximately 500, preferably 5 to100, mg/kg of body weight every 24 hours, if appropriate in the form ofseveral individual doses, to achieve the desired results. An individualdose contains the active compound(s) according to the inventionpreferably in amounts from approximately 1 to 80, in particular 3 to 30,mg/kg of body weight.

BIOLOGICAL TESTING

1. Growth Inhibition Test for the Determination of the CytotoxicProperties:

The human large intestine cell lines SW 480 and HT 29 (ATCC No. CCL 228and HBT 38) and the mouse melanoma cell line B16F10 (CRL 6475) weregrown in Roux dishes in RPMI 1640 medium with addition of 10% FCS. Theywere then trypsinized and taken up in RPMI plus 10% FCS to a cell countof 50,000 cells/ml for SW 480 and HT 29 and 20,000 cells for B16F10. 100μl of cell suspension/well were added to a 96 microwell plate andincubated for 1 day at 37° C. in a CO₂ incubator. A further 100 μl ofRPMI medium and 1 μl of DMSO containing the test substances were thenadded. The growth was checked after day 6. To do this, 25 μl of MTTsolution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)having a starting concentration of 5 mg/ml of H₂O were added to eachmicrowell. Incubation was carried out for 5 hours in a CO₂ incubator at37° C. The medium was then aspirated and 100 μl of i-propanol/well wereadded. After shaking for 30 min with 100 μl of H₂O, the extinction wasmeasured at 595 nm using a multiplate reader (Bio.) 3550 UV.

The cytotoxic action is indicated in Table 1 as the IC₅₀ value in eachcase for the SW 480 and HT 29 and B16F10 cell lines:

TABLE 1 IC₅₀/nM IC₅₀/nM IC₅₀/nM Example SW 480 HT 29 B16F1020(S)-Camptothecin  10  5  20 Example 1  70 40 200 Example 2 100 40 300Example 3 100 20 500 Example 4 100 20 500 Example 5  80 50 200 Example 6 60 30 300 Example 7  80 30  20 Example 8 200 100  800 Example 9 200 70400 Example 10 100 50 300

2. Haematopoetic Activity of the Glycoconjugate in Comparison with theUnderlying Active Compound:

Material and Methods:

Bone marrow cells were washed out of mice femurs. 10⁵ cells wereincubated at 37° C. and 7% CO₂ in McCoy 5A medium (0.3% agar) togetherwith recombinant murine GM-CSF (Genzyme; stem cell colony formation) andthe substances (10⁻⁴ to 100 μg/ml). 7 days later, the colonies (<50cells) and clusters (17-50 cells) were counted.

Results:

As shown in Tab. 2, the glycoconjugates investigated show a drasticallyreduced inhibition of the bone marrow stem cell proliferation comparedwith the underlying active compound.

TABLE 2 Inhibition of the CSF-induced proliferation of mouse bone marrowstem cells IC₅₀ [ng/mL] 20(S)-Camptothecin 0.05 Example 1 15 Example 230 Example 3 15 Example 4 15 Example 5 20 Example 6 10 Example 7 8Example 8 45 Example 9 15 Example 10 30

3. In Vivo Inhibition of Tumour Growth in the Nude Mouse Model

Material:

For all in vivo experiments for investigation of the inhibition oftumour growth, athymic nude mice (NMRI nu/nu strain) were used. Theselected large-cell lung carcinoma LXFL 529 was grown by serial passagein nude mice. The human origin of the tumour was confirmed byisoenzymatic and immunohistochemical methods.

Experimental Set-up:

The tumour was implanted subcutaneously into both flanks of small nu/nunude mice 6 to 8 weeks old. The treatment was started, depending on thedoubling time, as soon as the tumours had reached a diameter of 5-7 mm.The mice were assigned to the treatment group and the control group (5mice per group with 8-10 assessable tumours) by randomization. Theindividual tumours of the control group all grew progressively.

The size of the tumours was measured in two dimensions by means of aslide gauge. The tumour volume, which correlated well with the cellcount, was then used for all evaluations. The volume was calculatedaccording to the formula “length×breadth×breadth/2” ([a×b²]/2, a and brepresent two diameters at right angles).

The values of the relative tumour volume (RTV) were calculated for eachindividual tumour by dividing the tumour size on day X with the tumoursize on day 0 (at the time of randomization). The mean values of the RTVwere then used for the further evaluation.

The inhibition of the tumour volume (relative tumour volume of the testgroup/control group×100, T/C in %) was the final measured value.

Treatment:

The administration of the compounds was carried out intravenously(i.v.), for example on day 0, 1 and 2 after randomization, the totaldose per day being split over 2 administrations.

Results:

The therapeutic efficacy of the glycoconjugates according to theinvention from Examples 1 and 2 is shown by way of example in thelarge-cell human lung tumour xenograft LXFL 529. In the case of themaximum tolerable dose (MTD) and at ½ MTD, the therapy leads to completeto marked tumour remission. An excellent action can also be demonstratedon other tumours.

TABLE 3 Relative Number opt. body weight Dose Survival of T/C on day 7[% Therapy [mg/kg/day] time [days] tumours [%] of day 0] Control — 7 >28 8 100 104  >28 >28 >28 Example 1 16 >28 >28 10  0 95 >28 >28 (day28) >28 Example 1  8  0 >28 8 0 95 >28 >28 (day 14) >28 Example 232 >28 >28 8 0 98 >28  20 (day 21)  1 Example 2 16 >28 >28 8 3.3103  >28 >28 (day 28) >28

The long-lasting complete remission of the compound from Example 1 inthe dose range from 16 to 8 mg/kg and the dose-dependence of the actionis shown in a further experiment in FIG. 1 using the therapy scheduleday 1-3 i.v.

4. Hydrolytic Stability:

The compounds according to the invention from Examples 1, 2, 8 and 9 aredissolved in water and, after standing at room temperature for 24 h,show markedly less than 1% camptothecin release in the HPLC according toarea percent.

On dissolving 10 μM of the compounds from Examples 1 and 2 in RPMImedium plus 10% FCS and in 30% strength human whole blood in PBS buffer,only a camptothecin release of less than 5% took place after standingfor 24 h.

Method:

HPLC system Hewlett Packard HP 1050

Column: Nucleosil 120-5 C 18 250 mm×4 mm (Macherey & Nagel; Germany)

Eluent: A: 0.01 M KH₂PO₄ in H₂O (H₂=Milli-pore grade)

B: 80% acetonitrile/20% eluent A

Flow rate: 1.2 ml

Gradient: t₀: 20% B-t₄₀: 100% B

t₄₅: 100% B-t₄₇: 20% B

Detection: 240 nm or 370 nm

5. Lactone Stabilization:

The glycoconjugates according to the invention from Examples 1, 2 8 and9 are dissolved in 80% water and 20% of acetonitrile and adjusted to pH9 using 2 equivalents of sodium hydroxide solution. After standing atroom temperature for 1 h, the lactone ring opening is less than 5%(detection according to the above method).

EXAMPLES Carbohydrate Starting Material

I) p-Aminophenyl-3-O-methyl-β-L-fucopyranoside:

I.a) p-Nitrophenyl-3-O-methyl-β-L-fucopyranoside:

6 g (21 mmol) of p-nitrophenyl-β-L-fucopyranoside in 300 ml of absol.methanol are treated with 7.84 g (31.5 mmol) of dibutyltin oxide andheated under reflux for 2 h. The mixture is then concentrated, and theresidue is dried and then taken up in 300 ml of DMF. After addition of15.7 ml of methyl iodide, the batch is stirred at 70° C. for 40 h. Thesolvent is removed in vacuo and the residue is taken up in 300 ml ofdichloromethane. The suspension is filtered, and the remaining solutionis concentrated again and subjected to flash chromatography(dichloromethane/methanol 99:1). After concentration, 3.82 g (61%) ofthe target product are obtained.

I.) p-Aminophenyl-3-O-methyl-β-L-fucopyranoside:

3.81 g (12.73 mmol) of p-nitrophenyl-3-O-methyl-β-L-fucopyranoside aredissolved in methanol and, after addition of platinum dioxide,hydrogenated in a hydrogen atmosphere at a slight overpressure. Afterfiltering off the catalyst and precipitating with ether, 3 g (88%) ofthe target product are obtained. [TLC: dichloromethane/methanol 9:1R_(f)=0.53].

Peptidyl-camptothecin Starting Materials

II)20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-L/D-leucyl}-camptothecin,trifluoroacetate:

II) L20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-L-leucyl}-camptothecin,trifluoroacetate:

II) D20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-D-leucyl}-camptothecin,trifluoroacetate:

I.a) 20(S)-20-O-[N-(tert-Butoxycarbonyl)-L/D-leucyl]-camptothecin:

A suspension of 10 g (28.7 mmol) of 20(S)-camptothecin in 250 ml ofabsolute dimethylformamide is treated with stirring with 11.1 g (43mmol) of N-(tert-butoxycarbonyl)-leucine-N-carboxylic anhydride and 1 gof 4-(N,N-dimelthylamino)-pyridine. After treatment for 16 h in anultrasonic bath at room temperature, a further 3.7 g ofN-(tert-butoxycarbonyl)-leucine-N-carboxylic anhydride are added and themixture is left at room temperature for a further 2 h. It is thenseparated off from the residual undissolved camptothecin and thefiltrate is concentrated in vacuo. The residue is purified by flashchromatography [petroleum ether/ethyl acetate 1:1->1:4]. 13.55 g (84%)of the target compound IIa) are obtained. [TLC: acetonitrileR_(f)=0.47].

II.b) 20(S)-20-O-L/D-Leucyl-camptothecin, trifluoroacetate:

II.b) L 20(S)-20-O-L-Leucyl-camptothecin, trifluoroacetate:

II.b) D 20(S)-20-O-D-Leucyl-camptothecin, trifluoroacetate:

A solution of compound II.a (13.55 g, 24.1 mmol) in a mixture of 100 mlof dichloromethane and 40 ml of anhydrous trifluoroacetic acid isstirred at room temperature for 30 min. After concentrating in vacuo toa small volume, the product is precipitated with diethyl ether andthoroughly washed with diethyl ether. A double spot with R_(f) values of0.4 and 0.32 is detected in the thin-layer chromatogram(acetonitrile/water 20:1) [Yld.: 9.5 g (68%)].

By precipitating several times from dichloromethane/methanol usingdiethyl ether, the mixture can be separated into two individualcomponents II.b) L and II.b) D. Both forms prove to be diastereomerswhich produce slightly different ¹H-NMR spectra:

Polar diastereomer: 400-MHz-¹H—NMR (CD₂Cl₂/CD₃OD 1:1):δ s C—H (B-ring)8.63 ppm s C—H (D ring) 7.4 ppm Non-polar diastereomer: 400-MHz-¹H—NMR(CD₂Cl₂/CD₃OD 1:1):δ s C—H (B-ring) 8.60 ppm s C—H (D ring) 7.32 ppm

In the further stages, either the mixture of the two forms or thepurified diastereomerically pure forms can be employed.

II.c)20(S)-20-O-[N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxy-carbonyl)-L-lysyl-L/D-leucyl]-camptothecin:

II.c) L20(S)-20-O-[N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-lysyl-L-leucyl]-camptothecin:

II.c) D20(S)-20-O-[N^(α)-(tert-Butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-lysyl-D-leucyl]-camptothecin:

25.6 g (54.6 mmol) of[N^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-lysineand 11.1 g (82 mmol) of 1-hydroxy-1H-benzotriazole hydrate are dissolvedin 500 ml of dimethylformnamide. After addition of 12.6 g (1.2 eq.) ofN-ethyl-N′-(dimethylaminopropyl)-carbodiimide hydrochloride, the mixtureis stirred at room temperature for 1 h. Compound II.b (26.2 g, 0.83 eq.)and 7.83 ml (1 eq.) of ethyl-diusopropylamine are then added and thebatch is stirred at room temperature for a further 16 h. Afterconcentration in vacuo and purification by flash chromatography[petroleum ether/ethyl acetate 1:2->ethyl acetate], the target compound(43.5 g, 87%) is obtained [TLC: acetonitrile R_(f)=0.44].

As in the farther examples too, the batch can also be carried outcompletely analogously with each of the purified diastereomenrcally pureforms in II.b.

II)20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-L/D-leucyl}-camptothecin,trifluoroacetate:

II) L 20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-L-leucyl}-camptothecin,trifluoroacetate

II) D20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-D-leucyl}-camptothecin,trifluoroacetate

The compound II.c (43.3 g, 47.5 mmol) is taken up in 150 ml ofdichloromethane and treated with 50 ml of anhydrous trifluoroaceticacid. The resulting solution is stirred at room temperature for 1 h.After concentrating to a small volume in vacuo, the product isprecipitated by addition of diethyl ether. 39.4 g (90%) of the targetcompound are obtained [TLC: acetonitrile/water 10:1 R_(f)=0.35].

III) 20(S)-20-O-(L-Histidyl-L/D-valyl)-camptothecin, trifluoroacetate:

III.a) 20(S)-20-O-[N-tert-Butoxycarbonyl)-L/D-valyl)-camptothecin:

A suspension of 10 g (28.7 mmol) of 20(S)-camptothecin in 500 ml ofabsolute dimethylformamide is treated with stirring with 21.5 g (3equivalents) of N-(tert-butoxycarbonyl)-vatine-N-carboxylic anhydrideand 1 g of 4-(N,N-dimethylamino)-pyridine. After treatment for 16 h inan ultrasonic bath at room temperature, residual undissolvedcamptothecin is separated off and the filtrate is concentrated in vacuo.The residue is purified by flash chromatography [petroleum ether/ethylacetate 1:3->ethyl acetate]. 11.65 g (73%) of the target compound areobtained. {TLC: acetonitrile R_(f)=0.44]. If the same reaction iscarried out under reflux conditions in dichloromethane instead of indimethylformamide, the formation of the diastereomer with the Lconfiguration of the valine unit is strongly favoured.

III.b) 20(S)-20-O-L/D-Valyl-camptothecin, trifluoroacetate:

III.b) L 20(S)-20-O-L-Valyl-camptothecin, trifluoroacetate

III.b) D 20(S)-20-O-D-Valyl-camptothecin, trifluoroacetate

A solution of compound III.a (11.65 g, 21 mmol) in a mixture of 100 mlof dichloromethane and 100 ml of anhydrous trifluoroacetic acid isstirred at room temperature for 1 h. After concentrating in vacuo to asmall volume, the product is precipitated with diethyl ether and washedthoroughly with diethyl ether. The product is again precipitated fromdichloromethane/methanol with diethyl ether and isolated as a mixture ofdiastereomeric forms. Yld.: 10.9 g (92%) [TLC: acetonitrile/water 20:1R_(f)=0.31 and 0.39]. A greater purification of the non-polardiastereomer having the L-configuration of the valine unit is possiblein this stage by crystallization from methanol:

8 g of the resulting product having the enriched non-polar diastereomerare dissolved in 80 ml of methanol, cooled to 0° C. and treated withdiethyl ether in 10 ml steps. After addition of a total of 60 ml ofdiethyl ether, the precipitated product is filtered off with suction anddried. 4.6 g (58%) of pure non-polar diastereomer having theL-configuration of the valine unit are obtained. A reprecipitation ofthe mother liquor with diethyl ether affords a further 730 mg (9%) of aproduct fraction having a diastereomer ratio of 1:18.

In the further reactions, both the diastereomer mixture and the purifiednon-polar or the polar diastereomer can be employed. The reactionsproceed completely analogously.

III.c)20(S)-20-O-[N-tert-Butoxycarbonyl)-L-histidyl-L/D-valyl]-camptothecin:

III.c) L20(S)-20-O-[N-tert-Butoxycarbonyl)-L-histidyl-L-valyl]-camptothecin

III.c) D20(S)-20-O-[N-tert-Butoxycarbonyl)-L-histidyl-D-valyl]-camptothecin

5.95 g (23.3 mmol) of N-(tert-butoxycarbonyl)-L-histidine and 4.73 g of1-hydroxy-1H-benzotriazole hydrate are dissolved in 200 ml ofdimethylformamide. After addition of 5.38 g ofN-ethyl-N′-(dimethylaminopropyl)-carbodiimide hydrochloride, the mixtureis stirred at room temperature for 1 h. Compound III.b (10.9 g, 19.44mmol) and 6.7 ml of ethyl-diisopropylamine are then added and the batchis stirred at room temperature for a further 16 h. After concentrationin vacuo and purification by flash chromatography [acetonitrile/water50:1->20:1], the target compound is obtained [TLC: acetonitrile/water10:1 R_(f)=0.42], which is immediately reacted further.

III) 20(S)-20-O-(L-Histidyl-L/D-valyl)-camptothecin,bis-trifluoroacetate:

III) L 20(S)-20-O-(L-Histidyl-L-valyl)-camptothecin,bis-trifluoroacelate

III) D 20(S)-20-O-(L-Histidyl-D-valyl)-camptothecin,bis-trifluoroacetate

The compound III.c is taken up in 100 ml of dichloromethane and treatedwith 50 ml of anhydrous trifluoroacetic acid. The resulting solution isstirred at room temperature for 30 min. After concentrating to a smallvolume in vacuo, the product is precipitated by addition of diethylether. 13.05 g (83%) of the target compound are obtained [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.2].

IV)20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-D-tysyl-L/D-leucyl}-camptothecin,trifluoroacetate:

IV) L20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-D-lysyl-L-leucyl}-camptothecin,trifluoroacetate:

IV) D20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-D-lysyl-D-leucyl}-camptothecin,trifluoroacetate:

The synthesis of this compound is carried out in complete analogy to thediastereomeric compound II. In stage II.c, instead ofN^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-lysinethe corresponding D isomer is employed [TLC: acetonitrile/water 10:1R_(f)=0.4].

V)20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-ornithyl-L/D-leucyl}-camptothecin,trifluoroacetate:

V) L20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-ornithyl-L-leucyl}-camptothecin,trifluoroacetate

V) D20(S)-20-O-{N^(ε)-[Fluorenyl-9-methoxycarbonyl]-L-ornithyl-D-leucyl}-camptothecin,trifluoroacetate

The synthesis of this compound is carried out in complete analogy to thecorresponding lysine compound II. In stage II.c, instead ofN^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-lysineN^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-ornithineis employed. [TLC: acetonitrile/water 20:1 R_(f)=0.125].

VI) 20(S)-20-O-(L-Arginyl-L/D-leucyl}-camptothecin,tri-trifluoroacetate:

VI) L 20(S)-20-O-{L-Arginyl-L-leucyl}-camptothecin, tri-trifluoroacetate

VI) D 20(S)-20-O-{L-Arginyl-D-leucyl}-camptothecin, tri-trifluoroacetate

VI.a)20(S)-20-O-(Tri-tert-butoxycarbonyl-L-arginyl-L/D-leucyl)-camptothecin:

VI.a) L20(S)-20-O-{Tri-tert-butoxycarbonyl-L-arginyl-L-leucyl}-camptothecin

VI.a) D20(S)-20-O-{Tri-tert-butoxycarbonyl-L-arginyl-D-leucyl}-camptothecin

200 mg (0.42 mmol) of tri-tert-butoxycarbonyl-L-arginine and 80 mg of1-hydroxy-1H-benzotriazole hydrate are dissolved in 20 ml ofdimethylformamide. After addition of 97 mg ofN-ethyl-N′-(dimethylaminopropyl)-carbodiimide hydrochloride, the mixtureis stirred at room temperature for 1 h. Compound II.b (200 mg, 0.35mmol) and 217 μl of ethyl-diisopropylamine are then added and the batchis stirred at room temperature for a further 3 d. After concentrating invacuo and treating with water, the target compound is obtained [TLC:acetonitrile/water 5:1:0.2 R_(f)=0.77], which is immediately reactedfurther.

VI) 20(S)-20-O-{L-Arginyl-L/D-leucyl}-camptothecin,tri-trifluoroacetate:

VI) L 20(S)-20-O-{L-Arginyl-L-leucyl}-camptothecin, tri-trifluoroacetate

VI) D 20(S)-20-O-{L-Arginyl-D-leucyl}-camptothecin, tri-trifluoroacetate

0.35 mmol of the compound from VI.a) is stirred at room temperature for2 h with 5 ml of anhydrous trifluoroacetic acid in 10 ml ofdichloromethane. The mixture is concentrated and reprecipitated twicefrom dichloromethane/methanol using diethyl ether. 280 mg (82%) of thetarget compound are obtained [TLC: acetonitrile/water 10:3:1.5R_(f)=0.42].

VII) L20(S)-20-O-{N^(ε)[-Fluorenyl-9-methoxycarbonyl]-L-lysyl-L-valyl}-camptothecin,trifluoroacetate:

VII.a) L20(S)-20-O-[N^(α)-(tert-butoxycarbonyl)-N^(ε)-(fluorenyl-9-methoxycarbonyl)-L-lysyl-L-valyl}-camptothecin:

10.02 g (21 .4 mmol) ofN-(tert-butoxycarbonyl)-N-(fluorenyl-9-methoxycarbonyl)-L-lysine and 4.4g (32.1 mmol) of 1-hydroxy-1H-benzotriazole hydrate are dissolved in 400ml of dimethylformamide. After addition of 4.92 g (1.2 eq.) ofN-ethyl-N′-(dimethylaminopropyl)-carbodiimide hydrochloride, the mixtureis stirred at 0° C. for 30 min. 10 g (17.8 mmol) of the non-polardiastereomer III.b L of compound III.b and 9.2 ml (3 eq.) ofethyl-diisopropylamine are then added and the mixture is stirred at roomtemperature for a further 16 h. After concentrating in vacuo, theresidue is stirred with 500 ml of water for 30 min and filtered off withsuction. The product is taken up in 400 ml of dichloromethane, theresidual water is removed, and the solution is concentrated to 200 mland then precipitated with 800 ml of diethyl ether. The residue isfiltered off with suction and washed with diethyl ether. 14.712 g of thetarget compound are obtained (92%) [TLC: acetonitrile R_(f)=0.6].

VII) L20(S)-20-O-{N-[Fluorenyl-9-methoxycarbonyl]-L-lysyl-L-valyl}-camptothecin,trifluoroacetate:

The compound VII.a) L (14.65 g, 16.3 mnol) is taken up in 300 ml ofdichloromethane and treated with 50 ml of anhydrous trifluoroacetic acidat 0° C. The resulting solution is stirred with ice-cooling for 2 h.After concentrating to a small volume in vacuo, the product isprecipitated by addition of diethyl ether. 13.8 g (93%) of the targetcompound are obtained [TLC: acetonitrile/water 10:1. R_(f)=0.35].

Example 120(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-lysyl-L/D-leucyl}-camptothecin,hydrochloride

1.a)20(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[fluorenyl-9-methoxycarbonyl)-L-lysyl-L/D-leucyl}-camptothecin:

A solution of 9.82 g (36.5 mmol) ofp-aminophenyl-3-O-methyl-β-L-fucopyranoside (Example I) in 500 ml ofdioxane/water 1:1 is treated with stirring with 3.94 ml of thiophosgene(1.4 eq.). After 10 min, the mixture is treated with 4 equivalents ofethyldilsopropylamine, then concentrated in vacuo and the residue isdried for 1 h in an oil pump vacuum. The isothiocyanate obtained isdissolved in 500 ml of absolute dimethylformamide and treated with 30.4g (32.8 mmol) of compound II and 22.6 ml of ethyldulsopropylamine. Themixture is stirred at room temperature for 16 h, then concentrated invacuo and stirred with water. The residue is purified by flashchromatography [acetonitrile->acetonitrile/water 30:1]. The product isreprecipitated from dichloromethane/methanol using diethyl ether andwashed with diethyl ether. 28.7 g (78%) of the target product areobtained [TLC: acetonitrile/water 10:1 R_(f)=0.44].

1.b)20-O-{N^(α)-[-O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbony]-L-lysyl-L/D-leucyl}-camptothecin:

28.6 g (25.5 mmol) of the conjugate 1.a) are treated with 5 ml ofpiperidine in 200 ml of dimethylfonmamide. After stirring at roomtemperature for 2 h, the mixture is concentrated and the residue isdigested twice with dichloromethane and diethyl ether is added. It isthen taken up in dimethylformnamide/dichloromethane and precipitatedwith ether. This purification process is repeated twice. The product isfiltered off with suction and washed with ether. Yld.: 19.5 g (85%)[TLC: acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.3].

1)20(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-lysyl-L/D-leucyl}-camptothecin,hydrochloride:

10 g (11.1 mmol) of the compound from Example 1.b are taken up in 100 mlof water, treated with 11.1 ml (1 eq.) of a 1N HCl solution andlyophilized. The product is then precipitated several times fromdichloromethane/methanol using diethyl ether. Yld.: 9.15 g (88%) [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.3].

Example 220(S)-20-O-{N-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-histidyl-L/D-valyl}-camptothecin,hydrochloride

A solution of 7.14 g (26.5 mmol) ofp-aminophenyl-3-O-methyl-β-L-fucopyranoside (carbohydrate startingmaterial I) in 300 ml of dioxane/water 1:1 is treated with stirring with2.86 ml of thiophosgene (1.4 eq.). After 10 min, the mixture is treatedwith 4 equivalents of ethyldulsopropylamine, then concentrated in vacuoand the residue is dried for 1 h in an oil pump vacuum. Theisothiocyanate obtained is dissolved in 500 ml of absolutedimethylformamide and treated with 17.45 g (22 mmol) of compound III and22.7 ml of ethyl-diisopropylamine. The mixture is stirred at roomtemperature for 16 h, then concentrated in vacuo and the residue istaken up in water. It is adjusted to pH 7.8 using 1N aqueous sodiumhydroxide solution and the solid is filtered off with suction. Afterdrying in a high vacuum, the residue is reprecipitated twice fromdichloromethane/methanol using diethyl ether and washed with diethylether. 17.97 g (91%) of the target product are obtained, which is thenconverted into the hydrochloride using 1 equivalent of 0.1 N aqueoushydrochloric acid [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.36] [FAB-MS: m/e=896 (M+H⁺)].

Example 320(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-D-lysyl-D-leucyl}-camptothecin,hydrochloride

The synthesis is carried out completely analogously to Example 1. Thestarting materials I and IV.D are used as starting compounds here, thepolar diastereomer II.b) D being employed from the20-O-leucyl-camptothecin II.b stage. [TLC: acetonitrile/water/glacialacetic acid 5:1:0.2 R_(f)=0.31] [FAB-MS: m/e=901 M+H⁺].

Example 420(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-ornithyl-D-leucyl}-camptothecin,hydrochloride

The synthesis is carried out completely analogously to Example 1. Thestarting materials I and V) D are used as starting compounds here, thepolar diastereomer II.b) D having the D-leu configuration being employedfrom the 20-O-leucyl-camptothecin II.b stage. [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.25].

Example 520(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-arginyl-D-leucyl}-camptothecin

A solution of 73 mg (0.27 mmol) ofp-aminophenyl-3-O-methyl-β-L-fucopyranoside (starting material I) in 20ml of dioxane/water 1:1 is treated with stirring with 30 μl ofthiophosgene (1.4 eq.). After 10 min, the mixture is treated with 4equivalents of ethyldulsopropylamine, then concentrated in vacuo and theresidue is dried for 1 h in an oil pump vacuum. The isothiocyanateobtained is dissolved in 20 ml of absolute dimethylformamide and treatedwith 175 mg (0.18 mmol) of compound VI) D and 620 μl ofethyl-diisopropylamine. For the synthesis of compound VI) D, the polardiastereomer II.b) D is employed here from the 20-O-leucyl-camptothecinII.b stage. The mixture is stirred at room temperature for 16 h, thenconcentrated in vacuo and stirred with dichloromethane. The residue isthen reprecipitated from dichloromethane/methanol using diethyl etherand washed with diethyl ether. It is then lyophilized from dioxane/waterand then crystallized again from dichloromethane/methanol using diethylether. 154 mg (90%) of the target product are obtained [TLC:acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.39] [FAB-MS:m/e=929 M+H⁺].

Example 620(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fuopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-lysyl-D-leucyl}-camptothecin,hydrochloride

The compound is prepared in analogy to Example 1, the polar diastereomerII.b) D with the D-leucine configuration being employed in the20-O-leucyl-camptothecin II.b stage.

Alternatively, the compound from Example 1, for example, can also beseparated into the individual isomeric forms by preparative HPLC.

Conditions:

Separating column: Macherey & Nagel 250×21 mm Nucleosil 100-7 C18 AB

Eluent A: H₂O + 0.1M KH₂PO₄ Eluent B: Acetonitrile/water 4:1 + 0.02MKH₂PO₄ flow rate: 0 ml/min Inj. volume: 1500 μl Gradient 0-30% B 4-3020-90 22-90 24-30 Wavelength: 215 nm

After the HPLC separation, the corresponding fractions are lyophilizedand the residue is then precipitated several times fromdichloromethane/methanol using diethyl ether. The mixture is thenadjusted to pH 8-9, and the residue is filtered off and converted intothe hydrochloride using 0.1N hydrochloric acid.

In the ¹H-NMR spectrum, the isomers in Examples 6 and 7 show differentchemical shifts, in particular for the two singlets of the aromatic Hatoms in the camptothecin B ring or D ring.

Diastereomer with D-leucine

400-MHz-¹H-NMR (CD₂Cl₂/CD₃OD 1:1): δ s C—H (B ring) 8.52 ppm

s C—H (D ring) 7.42 ppm

[FAB-MS: m/e=901=M+H⁺]

Example 720(S)-20-O-{N^(α)-O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-lysyl-L-leucyl}-camptothecin,hydrochloride

The compound is prepared in analogy to Example 1, the non-polardiastereomer II.b) L with the L-configuration of leucine being employedin the 20-O-leucyl-camptothecin II.b stage.

Alternatively, the compound from Example 1, for example, can also beseparated into the individual isomeric forms by preparative HPLC.

Conditions: As Given in Example 6

After the HPLC separation, the corresponding fractions are lyophilizedand the residue is then precipitated several times fromdichloromethane/methanol using diethyl ether. The mixture is thenadjusted to pH 8-9, and the residue is filtered off and converted intothe hydrochloride using 0.1N hydrochloric acid.

In the ¹H-NMR spectrum, the isomers in Examples 6 and 7 show differentchemical shifts, in particular, for example, for the two singlets of thearomatic H atoms in the camptothecin B ring or D ring.

Diastereomer with L-leucine

400-MHz-¹H-NMR (CD₂Cl₂/CD₃OD 1:1): δ s C—H (B ring) 8.6 ppm

s C—H (D ring) 7.35 ppm

[FAB-MS: m/e=901=M+H⁺]

Example 820(S)-20-O-{N^(α)-[-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-lysyl-L-valyl}-camptothecin,hydrochloride

The compound is prepared in analogy to Example 1 in the non-polar seriesstarting from the compound VII) L.

The diastereomer ratio can be determined by analytical HPLC. Ifappropriate, a further purification of the diastereomer with theL-configuration of valine can be achieved by crystallization frommethanol (>20:1).

HPLC Conditions:

Separating column: Macherey & Nagel 250×4 mm Nucleosil 100-7 C18 AB

Eluent A: H₂O+0.1M KH₂PO₄

Eluent B: Acetonitrile/water 4:1+0.02M KH₂PO₄

flow rate: 1 ml/min

Inj. volume: 15 μl

In the ¹H-NMR spectrum, the pure diastereomer shows only one signal set,for example, for the two singlets of the aromatic H atoms in thecamptothecin B ring or D ring.

400-MHz-¹H-NMR (CD₂Cl₂/CD₃OD 1:1): δ s C—H (B ring) 8.55 ppm

s C—H (D ring) 7.35 ppm

[FAB-MS: m/e=887=M+H⁺]

An alternative procedure in the synthesis of the compound from Example 8is likewise possible. In this procedure, the carbohydrate unit fromExample 1 is first linked to a side chain-protected lysine derivative:

8a)N^(α)-[-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[fluorenyl-9-methoxycarbonyl]-L-lysine:

A solution of 6.8 g (25.3 mmol) ofp-aminophenyl-3-O-methyl-β-L-fucopyranoside (Example I) in 600 ml ofdioxane/water 1:1 is treated with stirring with 2.72 ml of thiophosgene(1.4 eq.). After 10 min, it is treated with 26 ml ofethyldiisopropylamine, stirred at RT for a further 5 min and thenconcentrated in vacuo to a volume of 150 ml. 800 ml of dichloromethaneare added and the phases are separated. The organic phase is washedtwice with water, dried over sodium sulphate and concentrated. Theresidue is stirred with 200 ml of methyl tert-butyl ether and 200 ml ofpetroleum ether and filtered off with suction. 7.26 g (92%) of theisothiocyanate are obtained.

2.92 g (9.4 mmol) of the isothiocyanate obtained are dissolved in 200 mlof dioxane/water 1:1 and treated with 3.11 g (0.9 eq.) ofN^(ε)-[fluorenyl-9-methoxycarbonyl]-lysine and 3.2 ml ofethyl-diisopropylamine. The mixture is stirred at room temperature for16 h, then concentrated in vacuo and the residue is taken up with water.By adjusting the pH to 2 with 1N HCl, the product is precipitated andfiltered off with suction after 30 min. The residue is suspended indichtoromethane and the solvent is stripped off twice. After washingwith ether and petroleum ether several times, 5.3 g (92%) of targetproduct are obtained [TLC: acetonitrile/water/glacial acetic acid5:1:0.2 R_(f)=0.69].

8b)20(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[fluorenyl-9-methoxycarbonyl]-L-lysyl-L-valyl}-camptothecin:

1.2 g (1.76 mmol) ofN^(α)-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-N^(ε)-[fluorenyl-9-methoxycarbonyl]-L-tysine(Example 8a) and 675 mg (1.2 mmol) of the compound III.b) L aredissolved in 50 ml of dimethylformamide, and the mixture is cooled to 0°C. and then treated with 346 mg (1.8 mmol) ofN-ethyl-N′-(dimethylaminopropyl)-carbodiimide hydrochloride. Thetemperature is allowed to rise to RT overnight with stirring. Thesolvent is evaporated in vacuo and the residue is stirred with water. Itis purified by flash chromatography on silica gel, starting withacetonitrile as eluent and later changing to acetonitrile/water 50:1.After concentrating the corresponding fractions, 1.06 g (76%) of thetarget product are obtained [TLC: acetonitrile/water 20:1 R_(f)=0.34].

The deblocking and the subsequent conversion into the hydrochloride iscarried out in analogy to Example 1.

Example 920(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-histidyl-L-valyl}-camptothecin,hydrochloride

Starting from the non-polar diastereomer of 20-O-valyl-camptothecin,trifluoroacetate (III.b) L, the compound is prepared in analogy toExample 2. The diastereomer ratio can be determined by analytical HPLC.If appropriate, a further purification of the non-polar diastereomerhaving the L-configuration of valine can be achieved by crystallizationfrom methanol (>20:1)

HPLC conditions:

Separating column: Macherey & Nagel 250×4 mm Nucleosil 100-7 C18 AB

Eluent A: H₂O+0.1M KH₂PO₄

Eluent B: Acetonitrile/water 4:1+0.02M KH₂PO₄

flow rate: 1 ml/min

Inj. volume: 15 μl

In the ¹H-NMR spectrum, the pure diastereomer shows only one signal set,e.g. for the two singlets of the aromatic H atoms in the camptothecin Bring or D ring.

Diastereomer with L-valine:

400-MHz-¹H-NMR (CD₂Cl₂/CD₃OD 1:1): δ s C—H (B ring) 8.58 ppm (overlaidby a CH arom. of histidine)

s C—H (D ring) 7.35 ppm

[TLC: acetonitrile/water/glacial acetic acid 5:1:0.2 R_(f)=0.36[[FAB-MS:m/e=896 (M+H⁺)].

Example 1020(S)-20-O-{N^(α)-[O-(3-O-Methyl-β-L-fucopyranosyl)-4-hydrdxy-phenylamino-thiocarbonyl]-L-arginyl-L-leucyl}-camptothecin

The product is prepared in analogy to Example 5. The non-polardiastereomer II.b) L is employed here in the 20-O-leucyl-camptothecinII.b) stage. [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2R_(f)=0.4[[FAB-MS: m/e=929=M+H⁺].

Using the HPLC conditions given under Examples 8 and 9, thediastereomers from Examples 5 and 10 can be differentiated.

What is claimed is:
 1. A camptothecin glycoconjugate of formula (I)

in which R¹ represents a β- or γ-branching non-polar amino acid sidechain; and R² represents a basic amino acid side chain; and their salts,stereoisomers and stereoisomer mixtures.
 2. The compound of formula (I)according to claim 1, where R¹ is a branched alkyl radical having up to4 carbon atoms; and R² is a radical of the formula —(CH₂)_(n)—R³, where

n is a number 1 to 4; and their salts, stereoisomers and stereoisomermixtures.
 3. The compound of the formula (I) according to claim 1, whereR¹ is a branched alkyl radical of formula

R² is a radical of the formula —(CH₂)₂—NH₂, —(CH₂)₃—NH₂, —(CH₂)₄—NH₂,

 and their salts, stereoisomers and stereoisomer mixtures.
 4. A processfor the preparation of a glycoconjugate of formula (I)

in which R¹ represents a β- or γ-branching non-polar amino acid sidechain; and R² represents a basic amino acid side chain, or of theirsalts, wherein the isothiocyanate of the formula (II)

 is reacted with the peptidyl-camptothecin, optionally bearing aprotective group in the side chain, of the formula (III)

 in which R¹ has the abovementioned meaning and R^(2′) has the meaningof the abovementioned basic radical R², which moreover can carry aprotective group on the basic group to give the glycoconjugate of theformula (IV)

 in which R¹ and R^(2′) have the meanings indicated above, wherein theside chain amino protective group which may be present is optionallyremoved and the compound obtained is optionally converted into thedesired salt.
 5. A process for the preparation of compounds of thegeneral formula (I) or of their salts, characterized in that theisothiocyanate of formula (II)

is reacted with an optionally protected terminal basic amino acid of theformula (V)

in which R^(2′) represents a basic amino acid side chain whose basicgroup can be protected, to give an amino acid conjugate of the formula(VI)

 in which R^(2′) has the meaning indicated above, this is then reactedwith amino acid conjugates of the formula (VII)

 in which R¹ has the meaning indicated in claim 1, wherein the sidechain protective group is optionally removed and the compounds areoptionally converted into a suitable salt.
 6. The compound20(S)-20-O-{N^(α)-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-lysyl-L-valyl}-camptothecinof the formula

and its salts.
 7. The compound20(S)-20-O-{N^(α)-[O-(3-O-methyl-β-L-fucopyranosyl)-4-hydroxy-phenylamino-thiocarbonyl]-L-histidyl-L-valyl}-camptothecinof the formula

and its salts.
 8. A pharmaceutical composition comprising a compoundaccording to claim 1 and a pharmaceutically acceptable carrier.
 9. Amethod for the treatment of oncoses, comprising administering to amammal in need thereof an effective amount of a compound according toclaim
 1. 10. The method of claim 9, wherein said mammal is a human. 11.A method of inhibiting tumor growth in a mammal, comprisingadministering an effective amount of a compound according to claim 1 tosaid mammal.
 12. The method of claim 11, wherein said mammal is a human.